Henry R. Hermann and Murray S. Blum.
The Hymenopterous Poison Apparatus. VI. Camponotus pennsylvanicus (Hymenoptera: Formicidae).
Psyche 75(3):216-227, 1968.

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THE HYMENOPTEROUS POISON APPARATUS.
VI. CAMPONOTUS PENNSYLVANICUS
(HYMENOPTERA: FORMICIDAE)
By HENRY R. HERMANN AND MURRAY S. BLUM
University of Georgia, Athens
METHODS AND MATERIALS
Workers of Ca~nponotus ftennsylvanicus were collected in Baton Rouge, La., in 1966 and
1967. Glandular and reservoir regions
were examined for morphological details after dissection of live specimens in normal saline. Sclerites were removed from the abdomen, dehydrated in ethyl alcohol, cleared in xylene and mounted in Permount. Some apparatuses were examined whole while the individual sclerites of others were disarticulated and examined indi- viduallv.
For preparations of histological sections, glands were removed in normal saline, dehydrated in ethyl alcohol, cleared in xylene and embedded in Paraplast. Tissue was sectioned at 10 microns, stained with Delafield's hematoxylin and eosin Y, and mounted on slides with Permount. All measurements for illustrations are in millimeters. In preparation [or chemical analysis, poison sacs were dissected from workers which had been relatively immobilized by storing them at 4OC for several hours. The glands were rinsed in distilled water and the venom was transferred to filter paper by puncturing the reservoir with a fine needle.
The venom-impregnated filter papers were subsequently treated with several diagnostic organic agents including Fehling's reagent, 2, 4-dinitrophenylhydrazine and ninhydrin. Ultimately a large series of compounds present in the poison gland secretion were re- solved by applying the contents of fifty glands to the origin of 141/?" X 13" sheets of Whatman # I filter papers. The venomous constitu- ents were analyzed by two-dimensional chromatography by employ- ing n-butanol : acetic acid : water (4 :I :I ) as the first phase ( I 7 hr) (Reed, 1950) and 80% aqueous pyridine as the second (8 hr) (Flavin and Anfinsen, 1954). The compounds were detected by spraying the papers with 0.2% ninhydrin in acetone following which the chromatograms were heated at IOOOC for 10 minutes. Standard mixtures of amino acids were run as controls in the same cabinet as the venom-treated paper and in some cases amino acid mixtures were co-chromatographed with the poison gland contents.



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19681 'Hermann and Blum - Camponotus 217 Much of the literature concerning the hymenopterous poison apparatus has been cited by Hermann a,nd Blum (1966, 1967a, 1967b). One of the first comparative investigations on the hymen- opterous poison apparatus that included descriptions on the apparatus of formicine ants was undertaken by Fore1 (1878). Since that time, Foerster (1912) contributed considerably to an understanding of the formicine poison apparatus, his work involving the skeletal and muscular components to describe a functional system. Emery ( 1922) and Buren ( 1944) noted that the nozzle-like pro- jection at the tip of the gaster of formicine ants is distinct from the cloacal orifice. As reviewed by Brown (1954), this projection forms a cone by an inrolling of the posterior portion of sternum VII and functions as a channel through which venom is sprayed to a con- siderable distance. This cone often has been misidentified as the cloacal orifice.
Hung and Brown (1966) dealt in detail with this nozzle-like structure in the Formicinae, calling it the acidopore. It was pointed out that the ring of fine setae surrounding the acidopore, the coronula, directs the venom spray outward away from the ant's body. The acidopore is situated on the ventral apex of sternum VII. In certain species of the Carnponotini, the acidopore may be formed as much by tergum VII as by sternum VII.
Carthy ( 195 I ) , Wilson ( 1963) and Blum and Wilson ( 1964) reported that the odor trail pheromone in certain formicine species was a product of the hindgut or some structure associated with the hindgut. Knowledge of this source of pheromone distinguishes this subfamily from some other subfamilies of ants in which the trail pheromone is a product of the poison gland (Blum and Moser, 1963; Blum et al., 1964), Dufour's gland (Wilson, 1959, 1962) or Pavan's gland (Wilson and Pavan, 1959).
Further anatomical investigations of the formicine poison apparatus were reported by Maschwitz ( 1964). In his work, Ma,schwitz de- scribed the poison sac and associated structures, Dufour's gland and the method of venom ejection in certain formicine species. We undertook the present investigation in part to describe the soft and sclerotized regions that make up the poison apparatus in Cam- ponotus pennsylvanicus ( DeGeer ). In an effort to characterize chemically the venoms of higher formicine ants, we undertook to identify the minor constituents that accompany formic acid in the venom of this species. At this juncture, we concentrated our efforts on establishing the chemical nature of the compounds secreted by the true poison gland in contradistinction to any components that may



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218 Psyche [September
be elaborated by Dufour's gland and ultimately mixed with the poison gland secretion. The venom produced by the poison glands of formicine ants has long been identified with formic acid, and it is quite clear that this compound is a consistent chemical denominator for the venomous secretions produced by members of the Formicinae. However, arthropod secretions usually have many components, and formicine venoms do not appear to violate this generalization. Thus, Stumper ( I 959) has demonstrated that the venom of Formica polyctena Forster contains in addition to formic acid, at least two other minor constituents. Similarly, Ghent ( 1961 ) reported that the venom of Camponotus pennsylvanicus (DeGeer) was character- ized by the presence of a water-soluble solid which constituted about 5% of the venom.
This research is part of a comparative study of the hymenopterous poison apparatus. An investigation of other formicine species is presently underway and will be reported on in forthcoming publica- tions.
RESULTS
The poison sac (PS) differs from the sacs of ants in other sub- families in that there is no gland invaginated into the sac (Fig. 2, A, B). Instead, an extremely long and narrow convoluted duct (CG) lies adnate to the dorsal surface of the sac. This duct branches into two long free filaments (FF) at the base of the sac. These filaments maintain a relatively uniform diameter throughout their length,
The basic composition of the reservoir, convoluted duct and free filaments is the same in this species as in other formicids previously described (Fore1 1878, Maschwitz 1964). The difference lies in the position of the convoluted region. In most formicids the con- voluted portion, responsible for enzymatic activity in changing pre- cursory compounds picked up by the free filaments, is totally within the sac (unpublished data). In C. pennsylvanicus, this region lies on the outside of the sac, and by pulling on the base of the free filaments, the elongate convoluted duct can be unraveled. Fig. 1. Comparison between the poison apparatus of CamPonotus penn- sylvanicus and what may be considered a typical stinging ant. A-Apparatus similar to that found in ponerine ants
(LV). B-Transverse section through
sting of a stinging ant species. C-Distal tip of sting showing barbed lancet tips extending posteriad from tip of sting shaft (LV). D-Poison apparatus of C. pennsylvanicus (LV).




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Hermann and Hum - Camponotus
poison sac spiraculor plate quadrate, plate Dufour's gland furfulo \ stinJ bulb
sting shaft
fulcra1 arm 1'
^
ing shaft




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2 20 Psyche [September
The sac is surrounded by a simple muscular layer which functions in forcing venom out of the sac. The convoluted region receives an abundant tracheal supply.
Dufour's gland (DG) in C. pennsylvanicus differs markedly from those glands in other subfamilies of ants. Instead of being an elongate unilobular sac, as is commonly found in ants previously examined, Dufour's gland in this species is bilobed (Fig. 2, C). A bilobed
Dufour's gland has been reported in Formica rufibarbis by Fore1 (1878) and in Formica polyctena by Maschwitz (1964). The components that make up the sclerotized portion of the poison apparatus (Fig. I, D) are basically similar to those in stinging Hymenoptera (Fig. I, A, B, C). However, some of the major sclerites have become reduced almost to a poin't beyond recognition. The oblong plate is relatively well developed (OP, Fig. I, D; Fig. 2, H) . However, its ramus (Ra 2) has been reduced consider- ably, so that now it is represented by a thin and slightly sclerotized bar only near the proximal end of the oblong plate. The fused second valvulae (sting) are wanting. Consequently, the levator muscle of the sting, normally originating on the posterior border of the second ramus and inserting on the anterior region of the sting bulb (SB, Fig. 2, G), is also wanting.
Although gonostyli are present (Go, Fig. I, D ; Fig. 2, I), they are membranous and possess minute setae (Set) along the lateral and ventrolateral regions.
Most stinging Hymenoptera possess long setae on each gonostylus (Fig. I, A), especially in the caudal and ventro- caudal region.
The first valvifers (triangular plates, TP, Fig. I, D; Fig. 2, E) are triangular in appearance, and each articulates anteriorly to a slender ramus (Ra I). In stinging Hymenoptera, each of the rami articulates ventrally with an elongate lancet shaft (LS) the latter usually terminating distally as a pointed and barbed structure (Fig. I, C). However, in C. pennsylvanicus each first valvifer is no longer a lancet shaft, but an elongate rod that terminates in a spatulate distal end (Fig. I, D; Fig. 2, E). The valve, a structure that Fig. 2.
Components of the poison apparatus of C. pennsylvanicus and a stinging ant species (LV). A-Transverse section of poison sac. B-Poison sac of C. pennsylvanicus (DV). C-Dufour's gland of C. pennsylvanicus (DV). D-Lancet of Paraponera clavata (LV). E-Lancet of C. pennsyl- vanicus (LV) . F-Spiracular plate of C. pennsylvanicus (LV) . G-Oblong plate, second ramus, gonostylus, fulcra1 arm and sting of P. clavata (LV). H-Oblong plate, gonostylus and portion of second ramus of C. pennsylwanicus (LV). I-Gonostylus of C. pennsylvanicus (LV).



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19681 di?er?nann and Blu?n - Camponot,us 22 I



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222 Psyche [September
functions in forcing venom through the sting shaft in stinging species (Va, Fig. 2, D), is lacking in C. fiennsyZvanicux. Each of the two quadrate plates (QP, Fig. I, D) is anatomically similar to those found in most stinging Hymenoptera (Fig. I, A). The distal region of each quadrate plate acts as a point of insertion for a muscle originating on the anterior and posterior regions of the oblong plate. The quadrate plate articulates anteroventsally with the dorsal apodeme of the triangular plate (TP). Neither fulcra1 (FA, Fig. I, A) arms nor a furcula (F'u) were found in this species. Both the fulcra1 arms and furcula articulate with the anterior end and antesoventral region of the sting bulb (SB, Fig. I, A) in stinging species. Since the sting bulb is wanting, at least 4 muscle groups that normally insert or have their origin on it are also wanting. In stinging forms, these muscles typically serve to deflect and rotate the sting, and pass over the poison canal to function as a sphincter in closing the passage through which venom issues during the act of stinging.
The spiracular plates (8th hemitergites, SP, Fig. 2, F) are basically the same as those in other hymenopterans (Fig. I, A). Since the general shape of these structures changes considerably throughout the Hymenoptera, it is difficult to discuss any significant differences be- tween this and other species at this point. Aside from formic acid, which constitutes nearly 50% of the volume of the poison gland secretion (Ghent 19611, the venom contains one other obvious constituent, a non-volatile residue. The existence of this water-soluble powder was noted by Ghent ( 1961 ) , who estimated that it represented about 5% of the whole venom. The residue does not have any pronounced odor and is relatively insoluble in organic solvents, especially those that are non-polar, A clue to the identities of at least some of the components in the residue was obtained after it was observed that the powdery deposit reacted intensely with ninhydrin. After analysis by two-dimensional chroma- tography, it became evident that the venom of C. pennsyZvanicus con- tains a large series of free amino acids. Thirteen amino acids were detected in the poison gland secretion. Based on the intensities of the colored ninhydrin-complexe,~, leucine, valine and serine appeared to be present in the highest concentrations. Lysine, proline, alanine, glutamine and x-aminobutyric acid were present in lower concentrations. Cystine, glycine, arginine, aspartic acid and threonine were minor constituents, A small amount of ninhydrin-positive material remained at the origin.



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19681 Hermann and BZum - Camponoi,us 223 DISCUSSION
Based on this investigation and descriptions of the poison apparatus of other formicine species, we can describe an apparatus that may be considered typical for the subfamily Formicinae. Without excep- tion, the poison apparatus of formicine ants, incIuding previously investigated species and several other species in our investigations not discussed here,
(I) is basically similar in appearance to that of stinging species, although (2) the gonapophyses that form the 2nd valvulae (sting bulb and shaft) are wanting; (3) there is no valve on the lancets; (4) the tongue-and-groove articulations between lancets and sting shaft have been lost;
(5) the fulcra1 arms are
wanting; and (6) the gonostyli have been reduced to membranous structures.
This apparatus of C. pennsyZvanicus is typical of formicines in other respects. The poison sac in all formicines investigated was large and possessed a convoluted structure on much of its dorsal surface. The free filaments extend from the base of the sac at the proximal end of the convoluted gland, Whether the poison sac is similar in aZZ formicine species will have to be investigated, although this form holds true for at least two species of Lusius and a species of Ac~nthomyops as well as several species of Cmnponotw and Formica.
Dufour's gland in C. pennsylvanicus is typical for species in the genera Cumponotus 2nd Formica that we have examined, but not for some of the more primitive genera. In some species of Lasius and Acuntho?nyops, Dufour's gland is distinctly unilobular. The presence of a large series of free amino acids in the venom of C. pennsylvanicus demonstrates for the first time, that formicine venoms share some common chemical characteristics with those of stinging ants. The majority of these amino acids are found in the poison gland secretion of the myrmicine Tetrmnorium guineense (I?.) (Blum and Ross 1965)~ and free amino acids are also charac- teristic of other myrmicine venoms (Blum 1966). Thus, although the venoms of no non-formicine ants are known to contain formic acid, the assumption that the venoms of formicine species share no common chemical components with those of stinging ants is no longer tenable.
The venom of C. pennsyZv~anicus may be typical of formicines in being fortified u7Xh free amino acids. We have examined also the venom of Formica paZZideJuZva Latreille and detected the presence of free amino acids. It is not improbable that the water-soluble residue



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224 Psyche [September
that Stumper (1959) noted in the venom of a member of the Formica rufa complex is simiIarly composed of free amino acids. It seems appropriate to ask what role, if any, free amino acids may play in enhancing the toxicity of the highly concentrated formic acid in the venom.
Ghent
(1961 ) has established that the formic acid in venom is spread over twice as large an area of the insect cuticle as the same concentration of control aqueous formic acid. He thus concludes that the white residue (amino acids) contributes to the toxicity of the formic acid by distributing the toxicant over a larger area than would be treated otherwise. However) it is worth bearing in mind that Stumper (1959) detected also an odorous constituent in the venom of a species of Formica. Stumper speculates that this volatile component may have arisen from the Dufour's gland secre- tion, thus introdccing the possibility that the secreted venom of formicines may contain products originating in two glands. In view of this distinct possibility, it is premature to attempt to explain the roles of poison gland products without considering the probably significant contribution to the toxicity of formicine venoms that the Dufour's gland products may make.
It should by no means be concluded that the chemistry of the formicine poison gland secretion is elucidated completely. The poison gland contents of C. pennsyZvanicus contain, in addition to the de- scribed compounds, three compo~mds that reduce aromatic amino salts after the ~oisun gland secretion has been subjected to thin layer chromatographic analysis. These compounds do not correspond to any amino acids, and they must represent unidentified constituents characteristic of the venom of this species. It may be no exaggeration to state that the elaborate formicine poison gland may yet be demon- strated to be a rich source of ~~nsuspected natural products. Although Carnponotus pennsyZvanicus has well defined defensive mechanisims of biting and introducing acid into the wound, or merely the spraying of acid and other substances, some of the sclerites that take part in stinging in more primitive formicids are markedly re- duced in this species. However, the glands and reservoir regions associated with the apparatus are well developed. The white residue in the dry poison gland secretion consists of a series of 13 amino acids. Leucine, valine and serine are the major free amino acids present. The chemistry of formicine venoms and the possible roles played by their constituents are discussed.



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LITERATURE CITED
BLUM) M. S.
1966. The source and specificity of trail pheromones in Termitopone, Monomorkm and Huberia, and their relation to those of some other ants. Proc. Roy. Ent. SOC. Lond. (A) 41: 155-160. BLUM) M. S.) J. C. MOSER AND A. D. CORDERO 1964. Chemical releasers of social behavior. 11. Source and specificity of the odor trail substances in four attine genera, (Hymenoptera: Formicidae). Psyche, 71 : 1-7.
BLUM) M. S. AND G, N. Ross
1965. Chemical releasers of social behavior, V, Source) specificity and properties of the odor trail pheromone of Tetramorium guineense (I?.) (Formicidae : Myrmicinae) . J. Insect Physiol. 11 : 857-868. BLUM) M. S. AND E, 0. WILSON
1964. The anatomical source of trail substances in formicine ants. Psyche 71 : 28-31.
BROWN, W. L.) Jr.
1956. Remarks on the internal phylogeny and subfamily classification of the family Formicidae) Ins. Sociaux 1: 21-31. BUREN) W. F.
1944. A list of Iowa ants, Iowa State Coll- Jour Sci. 18: 277-312. CARTHY) J. D.
1951. The orientation of two allied species of British ants. 11. Odor trail laying and following in Acantl~omyo$s (Lasius) fuliginosus. Behavior 3: 304-18.
EMERY) C.
1922. L'ouverture clocale des Formicinae ouvrieres et femelles. Bull. SOC. Ent. Belg. 4: 62-5.
FLAVIN) M. AND c, B. ANFINSEN
1954. The isolation and characterization of cysteic acid peptides in studies on ovalbumin synthesis. J. Biol. Chem, 211: 375-390. FOERSTER) E.
1912. Vergleichendanatomische Untersuchunge uber den Stechapparat der Ameisen. 2001. Jahrb. (Sect. 2). Abt. Anat, Ontog. Tiere. 34: 347-80.
FORBES) J.
1954. The anatomy and histology of the male reproductive system of CamPonotus pennsylwanicus DeGreer (Forrnicidae) Hymenoptera) J. Morph. 95: 523-47.
FOREL) A.
1887. Der Giftapparat und die Analdrusen der Ameisen. Zeitschr. f. wiss. 2001. 30: 28-66.
GHENT) R. L,
1961. Adaptive refinements in the chemical defense mechanisms of cer- tain Formicinae. Ph,D. thesis. Cornell University (unpublished). HERMANN) H. R.) JR., AND M. S. BLUM
1966. The morphology and histology of the hymenopterous poison ap- paratus, I. Paraponera clavata (Formicidae) . Ann. Entomol. SOC. Amer. 59: 397-409,




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226 Psyche [September
1967a. The morphology and histology of the hymenopterous poison ap- paratus. 11. Pogonomyrmex badius (Formicidae) Ann. Entomol. SOC. Amer. 60: 661-8.
1967b. The morphology and histology of the hymenopterous poison ap- paratus. 111. Eciton hamatum (Formicidae) . Ann. Entomol. Soc. Amer. 60: 1282-91.
HUNG, A. C. F., AND W. L. BROWN
1964. Structure of gastric apex as a subfamily character of the Formi- cinae (Hymenoptera : Formicidae) . N. Y. Entomol. SOC. 74: 198-200.
MASCHWITZ, U.
1964. Gefahrenatarmstoffe and Gefahrenalarmierung bei sozialen Hymenopteran. vergl. Physiol. 47 : 496-655. MOSER, J. C. AND M. S. BLUM
1963. Trail marking substance of the Texas leaf-cutting ant: Source and Potency. Science 140 :1228.
REED, L. J.
1950. The occurrence of x-aminobutyric acid in yeast extract: its iso- lation and identification. J. Biol. Chem. 183: 451-458. STUMPER, R.
1959. Un noveau constituent odorant du venin acide de fourmis. Compt. Rend. 249 : 1154-1156.
WILSON, E. 0.
1959. Source and possible nature of the odor trail of the fire ant Solenopsis saevissima (Fr. Smith). Science 129 : 643-44. 1962. Chemical communication amoung workers of the fire ant Solen- opsis saevissima (Fr. Smith). 1. The organization of mass- foraging. 2. An information and analysis of the odour trail. 3. The experimental induction of social responses. Animal Be- havior. 10: 134-64.
1963. The social biology of ants. Ann. Rev. Entomol. 8: 345-68. WILSON, E. O., AND M. PAVAN
1959. Source and specificity of chemical releasers of social behavior in the dolichoderine ants. Psyche 66: 70-6.



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CE
CG
DA
DG
DV
FA
FF
Fu
Go
Lbs
LS
LV
MD
OP
PS
QP
Ra 1
Ra 2
SB
Set
Sft
SP
SP
ss
TM
TP
Va
Hermann and BZum - Camtonotus
ABBREVIATIONS USED IN FIGURES
Columnar epithelium
Convoluted gland
Dorsal apodeme of oblong plate
Dufour's gland
Dorsal view
Fulcra1 arms
Free filaments
Furcula
Gonostylus
Lobes of Dufour's glands
Lancet shaft
Lateral view
Main duct of poison sac
Oblong plate
Poison sac
Quadrate plate
Ramus of first valvifer (triangular plate) Ramus of second valvifer (oblong plate)
Sting bulb
Setae on gonostylus
Shaft of lancet
Spiracle
Spiracular plate
Sting shaft
Transverse plate
Triangular plate
Valve of lancet




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